Dear all,
I am trying to harvest the low-expression plasmids in the mentioned E.coli strain. I constantly gel very low concentrations (30-50 ng/uL while the concentration is usually around 100 ng/uL for low-copy number plasmids) and poor quality: the 260/230 ratio is also very low (0.5-1.3 , normal=2.0-2.2). Thus, I cannot even Sanger-sequence them without a lot of ambiguity in the results as signal intensity is 20-300 with a normal signal intensity of no less than 700.
The optimal time and temperature for this strain are 12-16 hours and 37 °C to avoid plasmid loss. However, some people suggest propagating the low-copy number plasmids for no less than 20 hours. I have heard that after 16 hours of propagation, bacteria break down the antibiotic and get rid of the plasmids. I tried both LB and TB broths and TB gave a much denser cell pellet, however the plasmid concentration after DNA miniprep is the same as with LB. Both vectors were expressed under 100 mg/mL Kanamycin or Ampicillin. I propagated the cells in 10 mL TB with the mentioned antibiotics respectively to the selection marker for 13.5 hours at 225 rpm at 37 °C.
DNA Miniprep was done accordingly to the QIAGEN protocol with some changes. Instead of PB buffer wash, triple 80% EtOH washes with 1 min 13,000 rpm centrifuging were done. Columns were left to dry for 10 minutes in ambiance followed by centrifuging to remove any residual ethanol. DNA was extracted with three washes of 65°C MilliQ water (20 uL, 20 uL, 10 uL) with a 4-min soaking period before centrifuging. I checked plasmid concentrations with Nanodrop 2000 before Sanger sequencing and the results were terrible with both low concentrations and small 260/230 ratios.
I have expected that TB will boost the plasmid concentrations, although the results were no different from LB, if not worse. I wonder what could go wrong and how the concentrations and quality of the product could be improved for the better signal intensity of Sanger sequencing.
So far I have done about 100 DNA minipreps, but the quality worsens each time no matter the stocks, antibiotics and broth freshness, procedure accuracy, and protocol. Any suggestions on how to increase the low-copy number plasmid concentration and quality?
Please express any suggestions regarding the propagation of pET-1M and pMCSG7 vectors. Thanks.
Oftentimes the problem with increased cell numbers you get using richer medium (like TB) is that you get inefficient lysis and often lower yields. So when you are doing the cell lysis step (P2), make sure the lysate really becomes clear. Try it with 1-2 ml cells instead of 10 and see if that works.
Oftentimes the problem with increased cell numbers you get using richer medium (like TB) is that you get inefficient lysis and often lower yields. So when you are doing the cell lysis step (P2), make sure the lysate really becomes clear. Try it with 1-2 ml cells instead of 10 and see if that works.
Oftentimes the problem with increased cell numbers you get using richer medium (like TB) is that you get inefficient lysis and often lower yields. So when you are doing the cell lysis step (P2), make sure the lysate really becomes clear. Try it with 1-2 ml cells instead of 10 and see if that works.
Follow the protocol!
Use the PB buffer wash. Do not add extra washes, especially not with ethanol. You are washing away your plasmid.
Follow the protocol for the drying & removing ethanol steps. You are probably over-drying your column.
Elute in TE buffer, not water. Try warming the TE to 60*C to increase the yield. Elute once (or twice if you really want). Three times means you are diluting out the plasmic in more buffer.
Also, you can trying growing larger culture volumes. 10 ml is a tiny volume. Do 500 ml and make sure to grow in a large flask in a shaking incubator to allow for enough oxygen.
Then, use 1-2 ml for a mini prep. You'll know the results within an hour or so. If it works well, great! Do a few more mini preps.
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